1. Field of the Invention
This invention relates to compositions and methods for extracting and separating bio-active compounds and, more particularly, to novel compositions of bio-active compounds derived, isolated, and/or extracted from Fenugreek and methods for using said novel compositions for affecting homeostasis and metabolism in mammals, in addition to methods for manufacturing the same.
2. The Background Art
Fenugreek is one of the oldest medicinal herbs and has been found to be native to southeastern Europe, northern Africa, and western Asia, although it is widely cultivated in other parts of the world. Fenugreek is known technically as Trigonella foenum-graecum, a member of the family Fabaceae, and commonly referred to as Greek hay.
As appreciated by those skilled in the art, Fenugreek is a legume and typically grows between two to three feet tall with light green leaves and small white flowers. A Fenugreek seed pod may contain between ten to twenty small, flat, yellow-brown seeds. Typically, a plant seed is formed having a thick or hard outer coat called a testa and often referred to as a seed coat. The inner portion of the seed coat usually contains a plant embryo and a nutritive tissue called endosperm, which surrounds the embryo. As the Fenugreek seed embryo matures, it consumes endosperm. Fenugreek seeds often have a pungent aroma and may have a bitter taste, which is said to be similar to celery.
Fenugreek has long been used as a medicinal herb and culinary additive in both Asia and the Mediterranean. As used herein, a “bio-active compound” may be defined as any substance that has an effect on living tissue. It is believed that the seed of the Fenugreek plant contains many active compounds with pharmaceutical applications such as, for example, iron, vitamin A, vitamin B, vitamin C, phosphates, flavonoids, saponins, trigonelline, and other alkaloids. Fenugreek may be taken as a stomach tonic and as a treatment for abdominal ailments. Western scientific research has provided insight into the chemical analysis of Fenugreek seeds, together with the extraction of 4-hydroxyisoleucine from Fenugreek seeds, and has, accordingly, suggested some clinical utilities of Fenugreek.
Sir L. Fowden conducted research into the analysis of Fenugreek. He taught the isolation and purification of 4-hydroxyisoleucine from Fenugreek and claimed that it is the principal unbound amino acid contained in the Fenugreek seed. In addition to 4-hydroxyisoleucine, Fowden found that Fenugreek also contains gamma-aminobutyrate, ammonia, lysine, histidine, arginine, and at least four (4) additional unidentified compounds. (See, Fowden et al, Phytochemistry, 12:1707, (1973).) Further investigation of the prior art suggests that the amino acids found in Fenugreek seeds may have some nutritional value. (See, Sauvaire et al, Nutr Rep Int, 14:527 (1976).)
Spectrophotometry methods have also been taught by those skilled in the art in the analysis of steroid sapogenin content of Fenugreek seeds and such prior art methods may be generally used in an effort to determine the composition of subfractions of defatted Fenugreek. (See, Baccou et al, Analyst, 102:458 (1977); Ribes et al, Proc Soc Exp Biol Med, 182:159 (1986).) In addition, those skilled in the art have used chloroform in an effort to extract 4-hydroxyisoleucine from Fenugreek seeds. (See, Alcock et al, Phytochemistry, 28(7):1835 (1989).) It has been found, however, that chloroform is toxic and generally unacceptable as an extraction method under standards established by the food and drug industry.
Studies have also shown that the natural analogue of 4-hydroxyisoleucine is more effective as an antidiabetic agent than a synthetic version. There is, therefore, a suggestion that the therapeutic effects of 4-hydroxyisoleucine are best obtained from extracts of the Fenugreek seed. However, using Fenugreek seeds as a raw material source for a nutritional supplement presents some difficulties. For example, one such difficulty or disadvantage stems from the fact that a large dose of Fenugreek seeds is usually needed in order to obtain therapeutic and other nutritional effects. Patients or consumers are often unwilling to incorporate even de-fatted and de-bitterized seeds into their diet. As readily appreciated, mild gastro-intestinal upset may occur at higher doses with non-defatted seeds. In addition, due to the high fiber content of Fenugreek seeds, prolonged and high dosage amounts may result in adverse side effects such as flatus or diarrhea.
The binding capabilities of the fiber in Fenugreek seeds may also affect nutrient availability, particularly of minerals. As appreciated, external application of Fenugreek seed may result in undesirable skin reactions. Thus, it would be an advancement in the art to provide a method for obtaining bio-active and therapeutic compounds from Fenugreek seeds, such that undesirable side effects resulting from ingesting the seed or portions thereof can be avoided.
During the research investigations described herein, those skilled in the art developed crude methods for extracting 4-hydroxyisoleucine from Fenugreek seeds. These prior art methods and extraction techniques have primarily focused on obtaining a “high-purity” extract of 4-hydroxyisoleucine. For example, the extraction of 4-hydroxyisoleucine using adsorption chromatography is known in the art. Such prior art methods, however, tend to yield only small quantities of 4-hydroxyisoleucine and are typically only suitable for small scale laboratory use. As previously described, an alternative extraction method exercised by those skilled in the art uses a toxic organic solvent, such as chloroform, to extract 4-hydroxyisoleucine from Fenugreek seeds, whereby contemplating inherent disadvantages to the consumer.
As indicated above, other compounds have also been isolated from Fenugreek seeds. In addition to the major isomers (2S,3R,4S)-4-hydroxyisoleucine, minor isomers 4-hydroxyisoleucine, and amino acids (including, lysine, histidine, and arginine) have been isolated. Later studies have confirmed the presence of 4-hydroxyisoleucine in Fenugreek seeds in two diastereoisomers: the major one being the (2S,3R,4S) configuration, representing about ninety percent (90%) of the total content of 4-hydroxyisoleucine, and the minor one being the (2R,3R,4S) configuration. (See, Alcock, Phytochemistry, 28:1835 (1989).)
As appreciated by those skilled in the art, the major isomer (2S,3R,4S) is presently interesting with respect to experimental evidence indicating its ability to stimulate glucose-induced insulin secretion in micromolar concentrations through a direct effect on pancreatic beta cell stimulation in a glucose dependent manner. Moreover, 4-hydroxyisoleucine is able to interact and induce additive insulinotropic effects (i.e., stimulating or affecting the production and activity of insulin, only in the presence of supranormal glucose concentrations). (See, Sauvaire et al, Diabetes, 47:206 (1998).)
Investigation of the prior art also discloses clinical studies to investigate the use of subfractions of Fenugreek in conditions of hyperglycemia, glucosuria, and hyperlipidemia which have been performed on rats, dogs, and human pancreatic tissue. (See, Shani et al, Arch Intern Pharmacodyn Ther, 210:27 (1974); Ribes et al, Ann Nutr Metab, 28: 37 (1984); Valette et al, Athersclerosis, 50:105 (1984); Madar, Nutr Rep Int, 29:1267 (1984).)
As appreciated by those skilled in the art, clinical studies directed to conditions of hyperglycemia, as well as other conditions involving the metabolism of carbohydrates, have only investigated 4-hydroxyisoleucine as an effector of insulin-mediated or insulin-dependent pathways. The available prior art do not teach or suggest, however, Fenugreek and/or 4-hydroxyisoleucine compositions which may work synergistically or independently from insulin or insulin-mediated pathways. More particularly, there are no known prior art teachings or suggestions that 4-hydroxyisoleucine may affect the body by an insulin-independent mechanism. Stimulation of non-insulin mediated pathways may be desirous for targeting the utilization of carbohydrates in certain organ systems, (e.g., muscles, liver, etc.). Likewise, it may be desirous to avoid the general and/or systemic effects that may occur by stimulating the pancreas to produce and secrete insulin.
In addition, clinical studies conducted on Fenugreek have focused on investigating a specific subfraction of the Fenugreek seed (e.g., testa and endosperm) or, in the alternative, have focused on the specific effect of 4-hydroxyisoleucine in animals and humans with diabetes or a cholesterol disorder. Prior art directed to investigations of Fenugreek seed subfractions have failed to disclose or teach specific useful compositions.
Moreover, little or no attention has been given to the value of other bio-active compounds (e.g., free amino acids) present in Fenugreek seeds, especially in augmenting the hypoglycemic and hypercholesterolemic actions of 4-hydroxyisoleucine. At least one prior art teaches there is little or no additional utility to be gained by extracting anything other than 4-hyrdroxyisoleucine from Fenugreek seeds. Therefore, and as readily appreciated by those skilled in the art, a safer and more commercially practicable method for extracting compositions containing 4-hydroxyisoleucine and other bio-active components from Fenugreek is therefore needed. In addition, and as readily appreciated by those skilled in the art, novel compositions containing 4-hydroxyisoleucine for affecting the body by an insulin-independent mechanism are also needed.